Optimization of adsorption and desorption processes for His-tag enhanced green fluorescent protein

• Main Authors: cheng tzu-wei, 程子維
• Other Authors: Chang yu-kaung
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/42141992611925992105

碩士 === 明志科技大學 === 生化工程研究所 === 96 === The method development for direct capture of His-tag enhanced green fluorescent protein (His-EGFP) from unclarified E.coli homogenates using stirred fluidized bed (SFB) has been described. Cu(II), Zn(II), Ni(II) and Co(II) metal ion were evaluated in immobilized metal affinity chromatography (IMAC) for the recovery of His-EGFP. Immobilized Ni(II) chelating adsorbent was found to be the most effective for capture of His-EGFP. The adsorption characteristic of Ni(II)-STREAMLINE chelating adsorbent used in this work for total protein and His-EGFP in unclarified E.coli homogenates has been assessed by the measurements of equilibrium adsorption isotherm and uptake rate. Optimal conditions for the elution of His-EGFP were also investigated in packed bed experiments conducted with clarified E.coli homogenates. On the basis of the results, direct capture of His-EGFP from unclarified E.coli homogenates by SFB was carried out under the different loading volumes (50-200 ml). The purification results showed that the His-EGFP was directly recovered using 150 mM Imidazol buffer (pH 7) from unclarified E.coli homogenate with a purification factor of 2.04 and yield of 73.6% in the single step. Other contaminants were completely eluted by using 25 mM EDTA ( pH 7).